Molecular and functional characterization of olfaction genes in tsetse flies

描述（由申请人提供）： 本研究的总体目标是提供有关舌蝇媒介介导其嗅觉过程的分子过程的信息，使它们能够定位其宿主和其他对其生存和繁殖至关重要的资源。这些目标将通过在肯尼亚具有医学和兽医重要性的选定采采蝇物种（Glossina pallidipes、Glossina pallidipes和Glossina fuscipes fuscipes）中寻找和验证嗅觉反应基因来实现。我们提出了一个综合的和跨学科的方法：1）确定嗅觉反应基因的差异，在同域Glossina pallidipes和Glossina pallidipes在辛巴山和Glossina fuscipes在Mbita在肯尼亚和2）确定嗅觉反应基因的功能作用，在同域G。pallidipes和G. Shimba hills和G. F.在肯尼亚的姆比塔地区。我们假设这些果蝇中的嗅觉基因是同源的，差异表达的，并显着影响果蝇对不同气味的反应。将从各个部位对选定采采蝇种属的成虫进行采样，并从头部组织中提取RNA。我们将通过使用下一代测序技术来确定不同物种之间表现出的全球转录组水平的异质性来进行发现项目。从苍蝇的肠道内容物中获得的DNA将进行具有COI标记的PCR扩增，以从生命数据系统条形码（BOLD）数据库中确定推定的宿主物种。在来自不同物种的比较转录组分析之后，我们将通过qPCR在体外验证所选择的一组推定的嗅觉相关基因的存在和表达谱。通过RNAi基因沉默方法结合触角电位图和风洞技术，将进一步研究选定的推定嗅觉基因的功能作用，以确定这些基因在调节果蝇生理和行为中的作用。嗅觉相关基因及其在调节采采蝇嗅觉行为中的功能作用的全面信息的可用性将有助于进一步研发利用这些基因来控制采采蝇媒介。这项研究将主要在肯尼亚的国际昆虫中心进行。 生理生态学和肯尼亚农业研究所-锥虫病研究中心与保罗Mireji合作，伴随赠款是全球传染病（GID）D43 TW 007391（08/01/2008- 03/31/2013）。

DESCRIPTION (provided by applicant): The overall goal of this study is to provide information on molecular processes in tsetse vectors that mediate their olfactory processes, enabling them to locate their hosts and other critical resources for their survival and reproduction. The goals will be achieved by prospecting for and validating olfaction responsive genes in selected tsetse species (Glossina pallidipes, Glossina austeni and Glossina fuscipes fuscipes) of medical and veterinary importance in Kenya. We propose an integrated and interdisciplinary approach to: 1) Determine differences in olfactory responsive genes in sympatric Glossina pallidipes and Glossina austeni in Shimba hills and Glossina fuscipes fuscipes in Mbita in Kenya and 2) Determine functional roles of olfactory responsive genes in sympatric G. pallidipes and G. austeni in Shimba hills and G. f. fuscipes in Mbita districts in Kenya. We hypothesize that putative olfactory genes among these flies are orthologuous, differentially expressed and significantly influence responses of the flies to different odors. Adults of the selected tsetse species will be sampled from respective sites and RNA will be extracted from head tissues. We will undertake a discovery project by using next generation sequencing technologies to determine global transcriptome-level heterogeneities exhibited between the different species. The DNA obtained from the gut contents of flies will be subjected to PCR amplification with COI marker to determine the putative host species from Barcode of Life Data systems (BOLD) database. Following the comparative transcriptome analysis from the different species we will in vitro validate the presence and expression profiles of select set of putative olfaction related genes through qPCR. The functional role(s) of selected putative olfaction genes will be further investigated through RNAi gene silencing methodologies in conjunction with electro-antennogram and Wind-Tunnel techniques to establish the roles of these genes in modulating the physiology and behavior of the flies. Availability of comprehensive information on olfaction related genes, and their respective functional roles in modulating tsetse olfactory behavior will facilitate further R&D to exploit these genes to control tsetse fly vectors. This research will be done primarily in Kenya at International Centre of Insect Physiology Ecology and Kenya Agricultural Research Institute - Trypanosomiasis Research Center in collaboration with Paul Mireji with companion grant being Global Infectious Diseases (GID) D43 TW007391 (08/01/2008- 03/31/2013).